**5. Diagnostic and treatment**

The diagnosis of mycotoxicosis is a common challenge for veterinarians, because the mycotoxin-induced disease syndromes can easily be confused with other diseases caused by pathogenic microorganisms. The liver is the primary target organ of acute injury from AF ingestion in all species. Although it is difficult to prove that a particular disease outbreak was caused by a mycotoxin (CAST, 2003).

A diagnosis of mycotoxicosis is usually made by feed analysis and histopathology because clinical signs of aflatoxicosis can be nonspecific and confusing. Histologic evaluation of the livers of affected animals and analysis of the feed for mycotoxin content are crucial to confirm the clinical diagnoses. Histopathology signs as bile-duct hyperplasia, hepatocellular degeneration, fatty change of hepatocytes, and mononuclear-cell infiltration of the hepatic parenchyma were observed in broiler chickens fed 1 ppm AFs (Eraslan et al., 2006; Ortatali and Oguz, 2001).

In a 2005 research study, broilers were fed a combination of AFs and fumonisins. The livers of affected birds were enlarged, yellowish, friable, and had rounded borders (Miazzo et al., 2005).

The HE-stained tissue sections were characterized by multifocal cytoplasmatic vacuolation, with a variable location within hepatic lobes. Hepatocellular damage manifested by marked cytoplasmic vacuolation and pyknotic nuclei was reported in a 2006 study of rats administered 2 mg/kg body weight of AFB1 (Sakr et al., 2006).

Testing for mycotoxins in food and in the patient can be difficult because of variation in toxic concentration and the inconsistent production of toxins (LaBonde, 1995). A complete blood cell count, serum chemistry panel, and analysis of bile acids, ammonia, and urine help to rule out other causes of acute or chronic liver disease (e.g., infectious, neoplastic, chemical, drug-induced, congenital). Serum activity of hepatic enzymes (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase) is usually elevated. Serum ammonia and bilirubin concentrations are often increased. If bleeding disorders are found on clinical examination, determination of coagulation times may be helpful. If an animal has died, macroscopic findings may include generalized icterus, liver damage, ascites, widespread hemorrhage, and edema of the gallbladder (Bastianello et al., 1987).

Pet food companies report that even minor changes to color, odor or texture of a pet food that have no bearing on safety are frequently reported to increase complaints to the companies' consumer relations department. Except for overt moldiness, obvious rancidity, or visible inclusion of foreign materials, most incidents of pet food contamination are unlikely to be apparent on gross inspection. Thus, collection of samples for laboratory analysis may be indicated when the food is suspect. Proper handling of the sample as legal evidence may be critical if there is a possibility of a lawsuit at a later date (Miller and Cullor,

In submission of pet food samples suspected of contamination, effort should be made to improve the chances of detecting the possible contaminant. Vague references to "look for poison" on a sample submission form does not give much assistance. A tentative diagnosis, or at least a thorough description of clinical signs and laboratory findings, may give clues to the facility running the analysis on the suspected food as to which contaminants are likely

The diagnosis of mycotoxicosis is a common challenge for veterinarians, because the mycotoxin-induced disease syndromes can easily be confused with other diseases caused by pathogenic microorganisms. The liver is the primary target organ of acute injury from AF ingestion in all species. Although it is difficult to prove that a particular disease outbreak

A diagnosis of mycotoxicosis is usually made by feed analysis and histopathology because clinical signs of aflatoxicosis can be nonspecific and confusing. Histologic evaluation of the livers of affected animals and analysis of the feed for mycotoxin content are crucial to confirm the clinical diagnoses. Histopathology signs as bile-duct hyperplasia, hepatocellular degeneration, fatty change of hepatocytes, and mononuclear-cell infiltration of the hepatic parenchyma were observed in broiler chickens fed 1 ppm AFs (Eraslan et al., 2006; Ortatali

In a 2005 research study, broilers were fed a combination of AFs and fumonisins. The livers of affected birds were enlarged, yellowish, friable, and had rounded borders (Miazzo et al.,

The HE-stained tissue sections were characterized by multifocal cytoplasmatic vacuolation, with a variable location within hepatic lobes. Hepatocellular damage manifested by marked cytoplasmic vacuolation and pyknotic nuclei was reported in a 2006 study of rats

Testing for mycotoxins in food and in the patient can be difficult because of variation in toxic concentration and the inconsistent production of toxins (LaBonde, 1995). A complete blood cell count, serum chemistry panel, and analysis of bile acids, ammonia, and urine help to rule out other causes of acute or chronic liver disease (e.g., infectious, neoplastic, chemical, drug-induced, congenital). Serum activity of hepatic enzymes (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase) is usually elevated. Serum ammonia and bilirubin concentrations are often increased. If bleeding disorders are found on clinical examination, determination of coagulation times may be helpful. If an animal has died, macroscopic findings may include generalized icterus, liver damage, ascites, widespread hemorrhage, and edema of the gallbladder (Bastianello et al.,

and hence which analyses to conduct (Dzanis, 2008).

administered 2 mg/kg body weight of AFB1 (Sakr et al., 2006).

**5. Diagnostic and treatment** 

and Oguz, 2001).

2005).

1987).

was caused by a mycotoxin (CAST, 2003).

2000).

Histologically, varying degrees of liver damage are observed depending on the length of exposure to aflatoxins and their concentrations in the diet. Typical lesions in chronic and subacute cases are bile duct proliferation, varying degrees of fibrosis, hepatocellular fatty degeneration, and megalocytosis. Acutely poisoned dogs show massive fatty degeneration and centrilobular necrosis of the liver as well as widespread hemorrhage. In addition to liver lesions, renal proximal tubular necrosis is often present in dogs poisoned by aflatoxins. Confirmation of aflatoxicosis should include testing of the suspect feed source for aflatoxins (Trucksees and Wood, 1994).

Even if the feed is not visibly moldy, mycotoxins may be present. It is recommended to contact a veterinary diagnostic laboratory for sampling and shipping instructions. Some laboratories also offer testing of fresh liver for aflatoxin B1. Additionally, a liver biopsy may be useful in ruling out other etiologies of liver disease (Puschner, 2002).

Treatment for hepatic dysfunction is symptomatic and supportive (e.g., fluids, B-complex vitamins, glucose). In many cases, lactated Ringer's solution supplemented with potassium (20 mEq/L) is administered as a maintenance solution. In cases with hypoalbuminemia, administration of dextrose is recommended. Aflatoxicosis resulting in severe hepatic failure may lead to a hypocoagulable status, requiring correction with frozen plasma or whole blood. No antidote is available. The prognosis depends on the extent and severity of liver dysfunction. Monitoring serum biochemical parameters may help to evaluate the extent of liver damage. If liver damage is extensive, the prognosis is guarded to poor. Ammoniation and certain adsorbents are effective in reducing or eliminating the effects of aflatoxins in animals (Park et al., 1988; Puschner, 2002).

While there is no specific treatment for mycotoxicosis, birds that are at high risk of exposure may benefit from supplementation with glucomannans and organic selenium, which appear to decrease the hepatotoxic and CNS changes associated with exposure (Ergün et al., 2006; Dvorska et al., 2007). The best way to protect pet birds from exposure to mycotoxins is to feed only human-grade grain, corn, and peanut products; avoid spoiled foods; and store grain products in cool, dry places (Lightfoot and Yeager, 2008).

#### **6. Preventative strategies**

Such experiences have reaffirmed the need for manufacturers to devote extensive resources to documenting product quality. In many cases the processes already in place exceed the recognized standards within the industry. Nonetheless, most companies have increased the screening and sourcing control on ingredients used in pet foods. Regulatory standards are provided at several levels to ensure safety and adequacy of commercial products. In addition, the manufacture and regulation of pet foods is continually progressing forward, which should result in even more veterinary and consumer confidence in commercially manufactured foods (Anonymous: FDA, 2005; 2008, 2011).

A control program for mycotoxins from field to table should involve the criteria of an Hazard Analysis Critical Control Point (HACCP) approach which will require an understanding of the important aspects of the interactions of the toxigenic fungi with crop plants, the on-farm production and harvest methods for crops, the production of livestock using grains and processed feeds, including diagnostic capabilities for mycotoxicoses, and to the development of processed foods for consumption as well as understanding the marketing and trade channels including storage and delivery of foods to the consumer. A good testing protocol for mycotoxins is necessary to manage all of the control points for finally being able to ensure a

Aflatoxins in Pet Foods: A Risk to Special Consumers 69

commercial pet foods, impacted the entire pet food industry, affecting the confidence of veterinarians and owners. Long-lived, healthy consumers (pets) contribute to greater sales, so breakdowns in product quality can have catastrophic effect on profits or even company viability. More research is needed to better address the pet mycotoxin problem. Safety and efficacy of foods intended for animals are of prime interest to manufacturers because the health problems of pets are of a highly emotional concern, besides the pet food safety is the responsibility of the pet food industry. In the other hand, pet owners must care to store the animal's food at home with regard to avoid fungal contamination, putting the open bags in a clean and dry place, with aeration and protected against humidity from environment. The

Association of American Feed Control Officials –AAFCO (2007). Industry Statistics &

Food and Drug Administration- FDA (2005). Diamond Pet Food Recalled Due to Aflatoxin. In: *Recall – Firm Press Release*. Accessed december 19, 2010, Available from:

Food and Drug Administration- FDA (2007). Pet Food Recall (Melamine)/Tainted Animal

from: <http://www.fda.gov/oc/opacom/hottopics/petfood.html>. American Pet Products Association Inc. – APPA (2011). Industry Statistics & Trends. In: 2011

 <http://www.americanpetproducts.org/press\_industrytrends.asp>. Adams, C.L., Conlon, P.D. & Long, K.C. (2004). Professional and veterinary competencies:

*Journal of Veterinary Medical Education,* Vol. 31, pp. 66–71.

<www.fda.gov/Safety/Recalls/ArchiveRecalls/2005>.

*FAO/WHO/UNEP*.Tunis, Tunisia, March 1999.

Trends. In: *American Pet Products Association, Inc.,* Accessed january 09, 2011,

addressing human relations and the human–animal bond in veterinary medicine*.* 

Feed Updated. In: *Recalls & Withdrawals,* Accessed december 19, 2010, Available

 Pet Products Trend Report. Accessed january 16, 2011, Available from: <http://www.americanpetproducts.org/press\_industrytrends.asp 2009/2010>. American Pet Products Manufacturers Association – APPMA (2006). National Pet Owners

Survey 2005/2006. In: *APPMA News*, Accessed january 21, 2010, Available from: < http://www.americanpetproducts.org/newsletter/october2006/index.html>. Armbrecht B.H., Geleta J.N., Shalkop W.T. & Durbin C.G. (1971). A subacute exposure of beagle dogs to aflatoxin. *Toxicology and Applied Pharmacology*, Vol. 18, pp. 579–85. Asao, T., Buchi, G., Abdel-Kader, M.M., Chang, S.B., Wick, E.L. & Wogan, G.N. (1963). Aflatoxins B and G. *Journal of the American Chemical Society*, Vol. 85, pp. 1706–1707. Bailly J.D., Raymond I., Le Bars P., Leclerc J.L., Le Bars J. & Guerre P. (1997). Aflatoxine

canine: cas clinique et revue bibliographique. *Revue de Médecine Vétérinaire.*Vol. 148,

chromosomal aberration yields of bone marrow cells in male Chinese hamsters after a single i.p. injection of aflatoxin B1. *Mutation Research*, Vol. 244, pp. 189-195. Bastianello, S.S., Nesbit, J.W., Williams, M.C. & Lange, A.L. (1987). Pathological findings in a

natural outbreak of aflatoxicosis in dogs. *Onderstepoort Journal of Veterinary Research*,

foods and feeds. *Proceedings of Working document of the Third Joint* 

Bárta, M. Adámková, T. Petr & J. Bártová. (1990). Dose and time dependence of

Bhat, R. (1999). Mycotoxin contamination of food and feeds. Mycotoxin contamination of

shelf-life of commercial products must be observed, even at home.

**8. References** 

Available from:

pp. 907–14.

Vol. 54, pp. 635–40.

food supply free of toxic levels of mycotoxins (Richard, 2007). This system could be applied to prevent the risks of mycotoxins in animals by the pet food industry.

Conventional detection methods for AFB1 require trained personnel, a laboratory environment, expensive equipment and often several hours or days in analytical time. Several commercial rapid test kits for use in determining the aflatoxin concentration are present in market. These test kits are self contained and thus no additional equipment is required. The kit system provide all the necessary instructions to complete an analysis and it also enables visual evaluation of the results of grains samples on farm or at buying point. It is possible to detect AFB1 in cereals, nuts, spices and their derived products. Food samples are prepared for analysis by simply shaking the sample by hand in the presence of an extraction solution. However, the biggest challenge is the detection of minimum level of aflatoxin on feed or ingredients. But, a representative sample is essential, because aflatoxins can be concentrated in a few kernels that contaminate an entire load. A multi-level probe sampling at several sites and depths will give the best results. AOAC approved methods generally agree that an initial sample weight of 10 pounds (5 kilograms) is desirable (Byrne, 2008; Phillips, 2007).

Pet food amelioration is often considered a practical solution for mycotoxin contamination. Food processing techniques such as sieving, washing, pearling, ozonation, and acid-based mold inhibition can reduce the mycotoxin content of cereal grains. Dietary supplementation with large neutral amino acids, antioxidants, and omega-3 polyunsaturated fatty acids as well as inclusion of mycotoxin-sequestering agents and detoxifying microbes may ameliorate the harmful effects of mycotoxins in contaminated pet food. Amelioration of pet food, however, should be used as an additional safety factor but not to replace the sound application of risk and safety determination (Leung et al., 2006).

Sorption methods for the detoxification of aflatoxins are being studied and applied for the enterosorption and inactivation of aflatoxins in the gastrointestinal tract. Hydrated sodium calcium aluminosilicate (HSCAS) is a phyllosilicate clay commonly used as an anticaking agent in animal feeds. HSCAS tightly and selectively adsorbs aflatoxin and it has been shown to prevent the adverse effects of aflatoxins in various animals when included in the diet. Studies have also confirmed that HSCAS can alter the bioavailability of aflatoxin in dogs. HSCAS does not interfere with the utilization of vitamins and micronutrients in the diet and protects dogs fed diets with even minimal aflatoxin contamination. However, it does not protect animals against other mycotoxins. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the petfood industry further assurance of canine diet safety (Bingham et al., 2004).

Bingham et al. (2004) realized a crossover study, using six dogs randomly fed a commercial dog food (no-clay control) or coated with HSCAS (0.5% by weight) were subsequently administered a sub-clinical dose of aflatoxin B1. Diets were switched and the process repeated. The HSCAS-coated diet significantly reduced urinary aflatoxin M1 by 48.4% (+/-16.6 SD) versus the control diet. It was demonstrated that HSCAS protected dogs fed diets with even minimal aflatoxin contamination. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the pet food industry further assurance of canine diet safety.

#### **7. Conclusion**

It is known that mycotoxin contamination in pet food poses a serious health threat to pets and recent problems with contamination, while affecting only a small percentage of commercial pet foods, impacted the entire pet food industry, affecting the confidence of veterinarians and owners. Long-lived, healthy consumers (pets) contribute to greater sales, so breakdowns in product quality can have catastrophic effect on profits or even company viability. More research is needed to better address the pet mycotoxin problem. Safety and efficacy of foods intended for animals are of prime interest to manufacturers because the health problems of pets are of a highly emotional concern, besides the pet food safety is the responsibility of the pet food industry. In the other hand, pet owners must care to store the animal's food at home with regard to avoid fungal contamination, putting the open bags in a clean and dry place, with aeration and protected against humidity from environment. The shelf-life of commercial products must be observed, even at home.

#### **8. References**

68 Aflatoxins – Detection, Measurement and Control

food supply free of toxic levels of mycotoxins (Richard, 2007). This system could be applied to

Conventional detection methods for AFB1 require trained personnel, a laboratory environment, expensive equipment and often several hours or days in analytical time. Several commercial rapid test kits for use in determining the aflatoxin concentration are present in market. These test kits are self contained and thus no additional equipment is required. The kit system provide all the necessary instructions to complete an analysis and it also enables visual evaluation of the results of grains samples on farm or at buying point. It is possible to detect AFB1 in cereals, nuts, spices and their derived products. Food samples are prepared for analysis by simply shaking the sample by hand in the presence of an extraction solution. However, the biggest challenge is the detection of minimum level of aflatoxin on feed or ingredients. But, a representative sample is essential, because aflatoxins can be concentrated in a few kernels that contaminate an entire load. A multi-level probe sampling at several sites and depths will give the best results. AOAC approved methods generally agree that an initial

sample weight of 10 pounds (5 kilograms) is desirable (Byrne, 2008; Phillips, 2007).

application of risk and safety determination (Leung et al., 2006).

provide the pet food industry further assurance of canine diet safety.

(Bingham et al., 2004).

**7. Conclusion** 

Pet food amelioration is often considered a practical solution for mycotoxin contamination. Food processing techniques such as sieving, washing, pearling, ozonation, and acid-based mold inhibition can reduce the mycotoxin content of cereal grains. Dietary supplementation with large neutral amino acids, antioxidants, and omega-3 polyunsaturated fatty acids as well as inclusion of mycotoxin-sequestering agents and detoxifying microbes may ameliorate the harmful effects of mycotoxins in contaminated pet food. Amelioration of pet food, however, should be used as an additional safety factor but not to replace the sound

Sorption methods for the detoxification of aflatoxins are being studied and applied for the enterosorption and inactivation of aflatoxins in the gastrointestinal tract. Hydrated sodium calcium aluminosilicate (HSCAS) is a phyllosilicate clay commonly used as an anticaking agent in animal feeds. HSCAS tightly and selectively adsorbs aflatoxin and it has been shown to prevent the adverse effects of aflatoxins in various animals when included in the diet. Studies have also confirmed that HSCAS can alter the bioavailability of aflatoxin in dogs. HSCAS does not interfere with the utilization of vitamins and micronutrients in the diet and protects dogs fed diets with even minimal aflatoxin contamination. However, it does not protect animals against other mycotoxins. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the petfood industry further assurance of canine diet safety

Bingham et al. (2004) realized a crossover study, using six dogs randomly fed a commercial dog food (no-clay control) or coated with HSCAS (0.5% by weight) were subsequently administered a sub-clinical dose of aflatoxin B1. Diets were switched and the process repeated. The HSCAS-coated diet significantly reduced urinary aflatoxin M1 by 48.4% (+/-16.6 SD) versus the control diet. It was demonstrated that HSCAS protected dogs fed diets with even minimal aflatoxin contamination. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may

It is known that mycotoxin contamination in pet food poses a serious health threat to pets and recent problems with contamination, while affecting only a small percentage of

prevent the risks of mycotoxins in animals by the pet food industry.

Association of American Feed Control Officials –AAFCO (2007). Industry Statistics & Trends. In: *American Pet Products Association, Inc.,* Accessed january 09, 2011, Available from:

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**5** 

*Brazil* 

**Aflatoxin Contamination** 

 Eduardo Micotti da Gloria *University of Sao Paulo – ESALQ,* 

**Distribution Among Grains and Nuts** 

Grains (cereals and oilseeds) and nuts in general are subject to mold attack, in preharvest and postharvest. Among molds that can attack these foods *A. flavus*, and *A. parasiticus* are important because they can produce aflatoxins that are considered a potent natural toxin (Wild & Gong, 2010). Aflatoxin can be produced mainly by different *Aspergillus* species, but *Emiricella* and *Petromyces* have been reported as aflatoxin producers (Frisvad et al., 2005). Aflatoxin contamination has been reported for grains as corn, soya, wheat, rice, and cottonseed, and nuts such as peanuts, almonds, Brazil nuts, hazelnuts, walnuts, cashew nuts, pecans, and pistachio nuts (Fuller et al., 1977; Ayres, 1977; Moss, 2002; CAST, 2003; Gürses, 2006). Despite aflatoxin contamination having been observed in several foodstuffs, the contamination of maize, peanuts, and oilseeds can be considered, in terms of diet

Based on deleterious problems that aflatoxin can cause to human and animal health, some countries established a maximum concentration for aflatoxins in specific products. According to published data (Van Egmond, 2007), until 2003 one hundred countries had established legal limits for mycotoxins, and most of them regulated the aflatoxins presence

Several biotic and abiotic factors can determine fungal infection and growth, as well as aflatoxin production in preharvest. Temperature, water availability, plant nutrition, infestation of weeds, birds, and insects, plant density, crop rotation, drought stress, presence of antifungal compounds, fungal load, microbial competition, substrate composition, and mold strain capacity to produce aflatoxin are some important factors. The incidence of these factors is different in preharvest among plants and production areas of the same farm, among different farms of the same region and among different producer regions. Even among grains of the same ear or peanuts of the same pod the differences can occur. In postharvest, factors such as temperature, availability of water, oxygen, and carbon dioxide, insect and rodents infestation, incidence of broken grains or nuts, the cleaning of the product, toxigenic fungal load, microbial competition, antifungal compound presence, and substrate composition are important too. Transport, waiting time for drying, drying system (temperature and drying rate), and storage conditions can affect these factors during the postharvest period (Dorner, 2008; Diener et al.,

As a result of variable conditions that can occur during pre and postharvest, the aflatoxin contamination level among grains and nuts within the same lot can have an extremely

exposure, the most important worldwide (Benford et al. (2010).

1987; Campbell et al., 2006; Molyneux et al., 2007).

**1. Introduction** 

in food and feeds.

